Aim: To determine the prevalence of Helicobacter pylori (H. pylori) infection among symptomatic adults in a tertiary hospital in a peri-urban setting in Southwestern Nigeria. Methodology: It was a retrospective, cross-sectional study conducted in the Medical Microbiology Laboratory, Department of Medical Microbiology and Parasitology, Babcock University Teaching Hospital, Ilisan-Remo, Ogun State, Nigeria, between January 2022 and June 2024. The study involved a review of the medical microbiology laboratory records to evaluate the H. pylori stool antigen results of samples obtained from adults with gastrointestinal symptoms suggestive of H. pylori infection. Results: A total of 1061 adults were included in the study with females accounting for 56.2% of the participants. The age range of the participants was 18 – 95 years with a mean age of 34.59 + 15.51. The prevalence rate of H. pylori infection was 32.4% (344/1061). The infection was higher in females (50.6%) than in males (49.4%) and it was statistically significant (P = 0.011). However, a higher proportion (36.6%) of males had the infection than females (29.2%). The age group 21 – 30 years accounted for 23.3% of the H. pylori-positive participants while 37.6% of participants in the age group 41 -50 years were H. pylori-positive, however it was not statistically significant. Conclusion: H. pylori infection is prevalent among symptomatic adults in this locality. Hence, screening for H. pylori is recommended for detection, prompt treatment and eradication of H. pylori infection to prevent long-term complications.