Background: Leishmania infantum is a canine zoonotic vector-borne protozoan pathogen transmitted by phlebotomine sand flies, whereas Anaplasma platys is a bacterium most likely transmitted by ticks. While vertical transmission of L. infantum from pregnant bitches to their offspring has been documented, thus far no studies have explored the possibility of vertical transmission of A. platys in dogs. This study investigated the occurrence of vertical transmission of L. infantum and A. platys in sheltered dogs during the first half of gestation, in an area of southern Italy characterised by a high incidence of infection by both pathogens. Methods: The study population included 20 bitches (n = 10 pregnant, at 25–35 days of pregnancy; n = 10 nonpregnant), all subjected to ovariohysterectomy, which were examined for the presence of L. infantum and A. platys via cytological screening of bone marrow and whole blood samples. Infection by L. infantum and A. platys was also tested by immunofluorescence antibody test (IFAT) and quantitative real-time PCR (qPCR) targeting both pathogens. Selected tissue samples (n = 210) collected during surgical procedures from bitches and foetuses (n = 20) were assessed for the presence of L. infantum and A. platys by qPCR targeting a fragment of the kinetoplast minicircle DNA (kDNA) and the 16S rRNA gene, respectively. Results: Leishmania infantum DNA was not amplified from either uteri or ovaries from pregnant bitches or foetal tissue samples, whereas a subset of ovarian (n = 2) and uterine (n = 4) tissue samples from non-pregnant bitches were infected, with parasite loads of up to 3.09 × 10 and 7.51 parasite/PCR reaction, respectively. Conversely, uterine (n = 10) and ovarian (n = 8) tissues from both pregnant and non-pregnant bitches, together with a subset (n = 5) of foetal tissue samples were qPCR positive for A. platys. Leishmania infantum and A. platys nucleic acids were amplified from two uteri from nonpregnant bitches, with parasite loads of up to 2.32 × 10-3 and 2.05 parasite/per PCR reaction, respectively. Conclusions: Results from this study suggest that, in contrast to L. infantum, A. platys can be transmitted from pregnant dogs to their offspring during the first half of gestation. This hypothesis remains to be verified, for instance via direct observation of parasites in postpartum foetal tissues