Between July 2024 and July 2025, we collected spleen samples from 119 wild rodents captured across various zoogeographical regions of Gwangju City, Republic of Korea, to screen for major zoonotic pathogens, including Anaplasma, Babesia, Bartonella, Borrelia, Coxiella, Ehrlichia, and Rickettsia species. Of these, 13 samples (10.92%) tested positive for Bartonella spp. The positive samples were cultured on blood agar, resulting in eight bacterial isolates. Internal transcribed spacer (ITS) gene sequencing and phylogenetic analysis confirmed all Bartonella-positive samples as B. grahamii. Antibiotic susceptibility testing revealed that all B. grahamii isolates exhibited multidrug resistance (MDR) to ciprofloxacin, nalidixic acid, cefoxitin, streptomycin, and tetracycline, and showed intermediate resistance to ampicillin. In contrast, the isolates were susceptible to ceftazidime, cefotaxime, ceftriaxone, imipenem, gentamicin, amikacin, azithromycin, chloramphenicol, and trimethoprim/sulfamethoxazole. These findings provide essential baseline data on the presence and resistance patterns of B. grahamii in wild rodent populations inhabiting peri-urban environments. The detection of MDR Bartonella strains underscores a potential public health concern, particularly regarding zoonotic spillover via ectoparasites such as ticks. Sustained molecular surveillance and antimicrobial resistance monitoring are warranted to mitigate future zoonotic threats.