This study tested multiple methods for assessing microbial populations and identified bacteria detected on supermarket food-contact surfaces. The results for culture medium–based conventional methods, i.e., swabbing, direct count, sampling discs, and Petrifilm, showed that surface cleaning was satisfactory. However, direct epifluorescence microscopy (DEM) and bioluminescence detection of adenosine triphosphate (ATP) showed that surface cleaning was unsatisfactory. The surfaces of the fruit and vegetables section were the most contaminated, followed by the sections for cured meats and cheeses, fresh seafood, the aisles of the sections tested, meat, and yogurts and desserts. Likewise, significantly higher microbiological counts were observed on painted wood than on stainless steel, plastic, and painted epoxy surfaces (p < 0.05). Room-temperature food storage areas had higher bacterial counts than refrigerated areas (p < 0.05). Although no Salmonella spp., Listeria monocytogenes, or Staphylococcus aureus were detected, opportunistic pathogenic bacteria such as coagulase-negative staphylococci, Listeria grayi, and Pseudomonas spp. were isolated. Furthermore, the identification of the opportunistic and emerging foodborne pathogen Cronobacter sakazakii and the indicator of bacterial contamination Escherichia coli demonstrates potential risks to consumer health. Moreover, the Enterobacteriaceae count was low; however, the high total microbiological count detected by DEM indicates that cleaning and disinfection procedures should be improved. Compared to conventional methods, the use of DEM with stainless-steel disc surfaces as sampling support could increase the accuracy of detecting and enumerating microbial populations on surfaces by identifying residual bacteria after inadequate disinfection procedures.