Background: Nosocomial infections among neonates are responsible for 30–40% of death in resource constrained countries. Objective: Thisstudy aimed to detect isolated bacterial resistance genes and to assess chlorine gas values, which were emitted during using Didecyl dimethylammonium Chloride (DDAC) sterilizer in incubators of neonates at selected sites of Baghdad Hospitals. Materials and Methods: Cl₂ was detected by a portable sensor device, antibiotic susceptibility test was determined using disc diffusion and detection of resistance genes by PCR technique. Results: Presence of chlorine gas concentrations in the second and first sites during winter, which were higher than in summer and concentrations were highly significant (P< 0.0001) in second site and significant (P< 0.05) in first site. Third and fourth sites did not record any concentrations of chlorine. Pathogenic bacterial were isolated from these sites that included Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus, Escherichia coli, Kleibsiella pneumonia, Pseudomonas aurogenosa and Enterobacter. Antibiotic susceptibility test was done using disc diffusion method, which showed that all isolates were resistant to tested antibiotics specially to ampiclox and azithromycin (P<0.05). Detection of resistance genes by PCR showed Gram negative isolate possess various percentages of genes including TEM (88.9%), OXA-1(77.8%), CTX-M (72.2%), SHV (66.7%) and Gram positive isolate possess blaz (100%). aacA and mecA1 had sameresistance genes prevalent (45.5%).   Conclusion: Using DDAC a sterilizer showed effectiveness in eliminating bacterial presence. Therefore, effective disinfectants must be used to eliminate microbes.