Vector-borne agents and Toxoplasma gondii are common in cats, with many being zoonotic. The current study investigated the prevalence of selected infectious agents in cats from Johannesburg, South Africa, for which no published data exists. Whole blood and sera were obtained from 102 cats with a variety of disease conditions. Total DNA was extracted from the blood and assayed using PCR techniques for Mycoplasma haemofelis, Candidatus M haemominutum, Candidatus M turicensis, Bartonella species, Ehrlichia species and Anaplasma species. Enzyme-linked immunosorbent assays were used to detect IgG and IgM serum antibodies to T gondii and IgG serum antibodies to Bartonella species. Associations between test results, patient characteristics and haematological values were also evaluated. Overall, 56 cats (55%) were positive in one or more of the assays. Haemoplasma DNA was amplified from 26 cats [M haemofelis: four cats (3.9%); Candidatus M haemominutum from 22 cats (21.6%)] and Bartonella species DNA was amplified from eight cats [Bartonella henselae: five cats (4.9%); Bartonella clarridgeieae: three cats (2.9%)]; DNA of Ehrlichia species or Anaplasma species were not amplified. Of the cats, 24 (23.5%) were seropositive for Bartonella IgG and 18 (17.6%) were positive for T gondii IgM (12 cats), IgG (eight cats), or both (two cats). The study concluded that Bartonella species haemoplasmas and T gondii are common in client-owned cats in the region and the diagnosis of feline vector-borne agents and T gondii is difficult without the use of specific diagnostic tests, as there are minimal patient characteristics or haematological changes that indicate infection.