Aims: Probiotic lactobacilli have a great potential to produce antimicrobial compounds that inhibit and control the microbial pathogen growth. The antimicrobial and antifungal activities of two probiotic strains, Lactobacillus rhamnosus IMC 501 (R) and Lactobacillus paracasei IMC 502 (R), and their 1 : 1 combination, named SYNBIO (R), were studied using four different methods. Methods and Results: Using two modified streak methods and a well diffusion method, the inhibitory activity of the probiotics and their metabolites towards six Gram-positive, nine Gram-negative pathogenic bacterial strains and eight Candida strains was tested. Antagonistic effect of probiotic Lactobacillus strains was also investigated by coculturing assay highlighting a significant inhibition of most of the pathogens tested in this study. The combination SYNBIO (R) showed a microbicidal activity against most of the strains tested in the study. Conclusions: Compared with the control, most of the pathogenic bacteria and yeast were inhibited by all probiotic strains tested to various degrees. Significance and Impact of the Study: Screening Lactobacillus strains according to their activity in various environmental conditions could precede the clinical efficacy studies for adjunct treatment with probiotics in cure of different gastrointestinal and vaginal tract infections.