Background: This study explored the effects of hyperbaric oxygen therapy (HBOT) on hemogram, serum biochemistry and hemostatic variables in female dogs undergoing laparoscopic-assisted ovariohysterectomy (OVH). Materials: Thirty adult, mixed-breed, healthy female dogs were randomly divided into the following three groups: HBOT + SURG (exposed to two absolute atmospheres (ATAs) for 45 min followed by laparoscopic-assisted OVH), HBOT (exposed to two ATAs for 45 min) and SURG (laparoscopic-assisted OVH). Blood samples were collected at T0 (at the admission), at T1, 24 h after T0 (immediately after HBOT in the HBOT + SURG and HBOT groups, and immediately before anesthetic premedication in the SURG group), and at T2, 48 h after T0 (24 h after HBOT and anesthetic premedication). Methods: Assessments included erythrogram, leukogram, thrombogram, renal and hepatic serum biochemistry, prothrombin time (PT), activated partial thromboplastin time (APTT), buccal mucosal bleeding time (BMBT) and bloodstain area (BA) on hygroscopic paper collected at the BMBT. Results: Both the HBOT + SURG and SURG groups presented neutrophilia (p ≤ 0.0039) at T2 and an increase of ALP at T2 (p ≤ 0.0493), the SURG group presented an increase in leukocyte count at T2 (p = 0.0238) and the HBOT + SURG group presented a reduction in lymphocyte count at T2 (p = 0.0115). In the HBOT + SURG group, there was a reduction in PT and APTT in relation to the baseline value (p ≤ 0.0412). Conclusions: A session of HBOT at two ATAs for 45 min did not cause changes in the BMBT or BA in healthy female dogs. Some blood parameters investigated (neutrophil and lymphocyte count, ALP, PT and APTT) were affected by the use of HBOT.