Aims: To investigate critical electrical and biological factors governing the efficacy of pulsed light (PL) for the in vitro inactivation of bacteria isolated from the clinical environment. Development of this alternative PL decontamination approach is timely, as the incidence of health care-related infections remains unacceptably high. Methods and Results: Predetermined cell numbers of clinically relevant Gram-positive and Gram-negative bacteria were inoculated separately on agar plates and were flashed with < 60 pulses of broad-spectrum light under varying operating conditions, and their inactivation measured. Significant differences in inactivation largely occurred depending on the level of the applied lamp discharge energy (range 3 center dot 2-20 J per pulse), the amount of pulsing applied (range 0-60 pulses) and the distance between light source and treatment surface (range 8-20 cm) used. Greater decontamination levels were achieved using a combination of higher lamp discharge energies, increased number of pulses and shorter distances between treatment surface and the xenon light source. Levels of microbial sensitivity also varied depending on the population type, size and age of cultures treated. Production of pigment pyocynanin and alginate slime in mucoid strains of Pseudomonas aeruginosa afforded some protection against lethal action of PL; however, this was evident only by using a combination of reduced amount of pulsing at the lower lamp discharge energies tested. A clear pattern was observed where Gram-positive bacterial pathogens were more resistant to cidal effects of PL compared to Gram negatives. While negligible photoreactivation of PL-treated bacterial strains occurred after full pulsing regimes at the different lamp discharge energies tested, some repair was evident when using a combination of reduced pulsing at the lower lamp discharge energies. Strains harbouring genes for multiple resistances to antibiotics were not significantly more resistant to PL treatments. Slight temperature rises (< 4 center dot 2 degrees C) were measured on agar surfaces after extended pulsing at higher lamp discharge energies. Presence of organic matter on treatment surface did not significantly affect PL decontamination efficacy, nor did growth of PL-treated bacteria on selective agar diminish survival compared to similarly treated bacteria inoculated and enumerated on nonselective agar plates. Conclusions: Critical inter-related factors affecting the effective and repeatable in vitro decontamination performance of PL were identified during this study that will aid further development of this athermal process technology for applications in health care and in industry. Very rapid reductions (c. 7 log(10) CFU cm-2 within < 10 pulses) occurred using discharge energy of 20 J for all tested clinically relevant bacteria under study when treated at 8 cm distance from xenon light source. While no resistant flora is expected to develop for treatment of microbial pathogens on two-dimensional surfaces, careful consideration of scale up factors such as design and operational usage of this PL technique will be required to assure operator safety. Significance and Impact of the Study: Findings and conclusions derived from this study will enable further development and optimization of this decontamination technique in health care and in food preparation settings, and will advance the field of nonthermal processing technologies.