Background Salmonellosis remains an important public health problem globally. The disease is among the leading causes of morbidity and mortality in developing countries experiencing poor hygiene and lack of access to clean and safe water. There was an increase of 1.2% reported cases of Salmonellosis between 2015 and 2016 in Njombe region, Southern Highland of Tanzania. Nevertheless, little is known on the factors contributing to transmission of this disease in the region. This study was conducted to bring up to date the prevalence, antibiotic susceptibility and factors associated with Salmonella infection in-patient having gastrointestinal complaints. Methods A cross-sectional study was conducted from December 2017 to February 2018 among patients with gastrointestinal complaints at Njombe Regional Hospital. Stool samples were submitted for culture on Xylose Lysine Deoxycholate Agar, intended for isolation of Salmonella spp. Identification was based on conventional biochemical tests and sero-typing. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disc diffusion method. Factors associated with occurrence of Salmonella infection were determined by regression analysis using Epi Info software version 3.5.4. Results The prevalence of Salmonella infection among 297 study participants with gastrointestinal features was 16.5%. All isolates were sensitive to ceftriaxone and ciprofloxacin, whereas 27.8% and 100% were resistant to co-trimoxazole and ampicillin respectively. The odds of Salmonella infection was fourfold higher among participants with formal employment than others (AOR=3.8, 95% CI, 1.53-9.40). Use of water from wells or river (AOR 2.2, 95% CI, 1.07-4.45), drinking untreated water (AOR 2.6, 95% CI, 1.21-5.48) and often eating at restaurant (AOR 3.4, 95% CI, 1.28-8.93) had increased probability of Salmonella infection. Likewise, having abdominal pain (AOR 8.5, 95% CI 1.81-39.78) and diarrhea (AOR 2.3, 95% CI 1.12-4.68) were independently associated with Salmonella infection. Conclusion Salmonella infection in Njombe is high, contributed by often eating at restaurant and drinking unsafe water from well and river. In addition, Salmonella spp causing gastroenteritis has developed resistance to commonly used antibiotics. It is high time to emphasize on hygienic practices in order to control and prevent Salmonella infection.