Bartonella henselae (B. henselae) is a zoonotic bacterium for which domestic cats serve as the primary reservoir host, maintaining persistent bacteremia and facilitating transmission mainly through the cat flea, Ctenocephalides felis. Given the public health significance of feline infection and the limited molecular epidemiological data available from southwestern Iran, this study aimed to determine the molecular prevalence of B. henselae and identify independent epidemiological risk factors associated with infection in owned and non-owned cats in Ahvaz, Iran. A cross-sectional study was conducted involving 160 cats, equally distributed between owned and non-owned populations. Adult cats constituted 65% of the study population, whereas juveniles accounted for 35%; 55% were male and 45% were female. Domestic Shorthair cats represented the predominant breed type (51.9%). Whole-blood, oral-swab, and nail samples were collected and analyzed using conventional polymerase chain reaction (PCR) targeting a 414-bp fragment of the htrA , followed by sequencing confirmation of representative amplicons. Associations between potential risk factors and infection were assessed using univariable and multivariable logistic regression analyses, and population attributable fractions (PAFs) were estimated. B. henselae DNA was detected in 16.9% of cats (95% CI: 11.4–23.7). The prevalence of infection was significantly higher in non-owned cats (26.3%) than in owned cats (7.5%) (OR = 4.39, 95% CI: 1.66–11.58; p = 0.002). Population attributable fraction analysis indicated that 67% and 36% of infections were attributable to outdoor exposure and flea infestation, respectively. These findings demonstrate that environmental exposure and ectoparasite infestation are major determinants of B. henselae infection, highlighting the importance of effective flea control and restricting outdoor access to reduce the burden of infection and mitigate zoonotic risk.