Background Microorganisms' survival is based on the importance of growth factors found in the microbial environment, as well as their overwhelming appetite for survival which is controlled by their genetic material. This study was designed to investigate the virulence patterns of Salmonella enterica serovar typhimurium associated with soft cheese. Two hundred and sixty (260) soft cheese samples were collected from vendors at Akure, Ikare and Owo in Ondo State, Nigeria. Molecular characterization of six (6) Salmonella enterica isolates for the possession of iro B, stn, hil A and spv C gene were revealed through multiplex polymerase chain reaction (PCR) analysis with DNA extraction, polymerase chain reaction (PCR) amplification, sequencing and genome blasting, respectively. Results Four (4) possess only iro B; two (2) possess only stn gene; and another three (3) possess hil A gene as none of the isolates possess spv C gene. This study revealed antibiotic-resistant Salmonella enterica serovar typhimurium is present in locally made soft cheese samples. It also revealed that virulence genes comprising hil A, stn and iro B as well as plasmids-mediating antibiotic resistance are present in S. enterica serovar typhimurium found in locally produced cheese. Conclusions The presence of molecularly elucidated virulence genes comprising Iro B, Stn and Hil A makes the soft cheese potentially viable for pathogenicity. This study recommends food-borne salmonellosis should be constantly monitored with appropriate cleaning of preparatory paraphernalia in cheese production. Constant sensitization of nomads to improve their awareness on milk-borne zoonosis and its associated risk factors is needed.