The effect of the surfactants polyoxyethylene monostearate (Tween 60), polyoxyethylene monooleate (Tween 80), cetyl trimethyl ammonium bromide (CTAB), and sodium dodecyl sulfate (SDS) on the estimation of bacterial density (sulfate-reducing bacteria [SRB] and general anaerobic bacteria [GAnB]) was examined in petroleum samples. Three different compositions of oil and water were selected to be representative of the real samples. The first one contained a high content of oil, the second one contained a medium content of oil, and the last one contained a low content of oil. The most probable number (MPN) was used to estimate the bacterial density. The results showed that the addition of surfactants did not improve the SRB quantification for the high or medium oil content in the petroleum samples. On other hand, Tween 60 and Tween 80 promoted a significant increase on the GAnB quantification at 0.01% or 0.03% m/v concentrations, respectively. CTAB increased SRB and GAnB estimation for the sample with a low oil content at 0.00005% and 0.0001% m/v, respectively.