The purpose of this study was to investigate the effect of chronic feline immunodeficiency virus (FIV) infection, and efficacy of marbofloxacin treatment, on Mycoplasma haemofelis infection. Six cats chronically infected with FIV-Glasgow8 (Group X) and six FIV-free cats (Group Y) were infected with M. haemofelis on Day 0 by intravenous blood inoculation. From Day 0 until Day 86 post-infection (pi), blood samples were collected for M. haemofelis and FIV provirus quantitative real-time PCR and haematology. Three of the six cats in each of Groups X and Y were randomly selected to receive marbofloxacin treatment (2 mg/kg PO q24 h) from Day 16 to 43 pi, with the remaining cats being untreated controls with no antibiotic treatment. The M. haemofelis copy numbers and haematological data were compared between Groups X and Y, and between marbofloxacin-treated and control cats using a Mann-Whitney U-test. M. haemofelis infection was associated with development of macrocytic hypochromic anaemia. In some cats, marked variation in M. haemofelis copy number over time (>100,000-fold difference within 48 h in some cats) and/or cycling of copy number was seen. No correlation was found between FIV provirus copy number and M. haemofelis copy number or haematological variables. No significant effect of chronic FIV infection on M. haemofelis copy number kinetics or haematological changes due to M. haemofelis infection was found, other than MCHC (P=0.03). Marbofloxacin treatment was associated with a significant decrease in M. haemofelis copy number (P=0.002), although consistent clearance of infection was not demonstrated. This study reveals the presence of marked fluctuations in M. haemofelis copy number kinetics in vivo and a significant response to marbofloxacin antibiotic treatment.