Autoimmune diseases (Ads) can be caused by genetics, gut microbiome, hormonal changes, environmental triggers, and viruses. The association between cytomegalovirus (CMV) infection and development of antinuclear antibodies (ANA) is controversial. Therefore, this study aims to investigate the potential connection between ADs and CMV infections in Jordanian patients. This observational comparative retrospective study included 214 participants divided into three groups: 113 with ANA-positive serum, 86 with ANA-negative serum, and 15 with positive autoantibodies. ZEUS IFA ANA HEp-2 test system was used to test for ANA and ARCHITECT kit for CMV IgG and IgM. The mean age of total patients was 41.31 ± 20.045 years. The majority of patients were females (58.9%) and from Amman city (73.8%). ANA positivity was detected in 22.1%, and skin disorders were found in 8.4% of total patients. The mean value of CMV IgG and CMV IgM antibodies were 206.65 ± 75.38 U/ml and 0.42 ± 1.09 U/m, respectively. Speckled, nucleolar, and centromere patterns of ANA were detected among 64.6%, 22.1%, and 6.2%, respectively of positive ANA patients. Age correlated positively with the presence of CMV-G antibodies (r = 0.446, p < 0.001), presence of disease (r = 0.428, p = 0.001), and type of disease (r = 0.264, p = 0.005). ANA titre correlated positively with the type of disease (r = 0.314, p = 0.001) among positive ANA patients. The results of this research could not establish a solid association between CMV-G and CMV-M antibodies and the general presence of ANA.