Antibiotics discovery was a significant breakthrough in the field of therapeutic medicines, but the over (mis)use of such antibiotics (in parallel) caused the increasing number of resistant bacterial species at an ever-higher rate. This study was thus devised to assess the multi-drug resistant bacteria present in sanitation-related facilities in human workplaces. In this regard, samples were collected from different gender, location, and source-based facilities, and subsequent antibiotic sensitivity testing was performed on isolated bacterial strains. Four classes of the most commonly used antibiotics i.e., β-lactam, Aminoglycosides, Macrolides, and Sulphonamides, were evaluated against the isolated bacteria. The antibiotic resistance profile of different (70) bacterial strains showed that the antibiotic resistance-based clusters also followed the grouping based on their isolation sources, mainly the gender. Twenty-three bacterial strains were further selected for their 16s rRNA gene based molecular identification and for phylogenetic analysis to evaluate the taxonomic evolution of antibiotic resistant bacteria (ARB). Moreover, the bacterial resistance to Sulphonamides and beta lactam was observed to be the most and to Aminoglycosides and macrolides as the least. Plasmid curing was also performed for multidrug resistant (MDR) bacterial strains, which significantly abolished the resistance potential of bacterial strains for different antibiotics. These curing results suggested that the antibiotic resistance determinants in these purified bacterial strains are present on respective plasmids. Altogether, the data suggested that the human workplaces are the hotspot for the prevalence of MDR bacteria and thus may serve as the source of horizontal gene transfer and further transmission to other environments.