A cross-sectional study was conducted on 274 cats in Southern Brazil to estimate the prevalence of Mycoplasma haemofelis PCR, associated factors, and its correlation with ELISA for FeLV and FIV. The apparent prevalence of M. haemofelis was 6.6% (18/274) (95% CI: 3.6–9.5%), of which 33.3% (6/18) had co-infection with FeLV, 5.6% (1/18) with FIV, and 5.6% (1/18) with both. Male cats were more likely to be positive for M. haemofelis [OR: 7.07 (1.97–25.34)]. Only three M. haemofelis-positive cats showed related clinical changes, such as mucosal pallor. A statistically significant difference was observed between M. haemofelis-positive cats and the negative control group for age, hemoglobin concentration, packed cell volume, and rod neutrophil counts. Mycoplasma haemofelis is prevalent in southern Brazil, with a higher risk in male cats. Most cats could be classified as asymptomatic carriers since they were healthy.