Background: Salmonellosis remains an important public health problem globally. The disease is among the leading causes of morbidity and mortality in developing countries that experience poor hygiene and lack of access to clean and safe water. There was an increase in reported cases of Salmonellosis in Njombe Region, Southern Highland of Tanzania between 2015 and 2016 based on clinical diagnosis. Nevertheless, little is known about the factors contributing to the transmission of this disease in the region. This study was conducted to determine the prevalence, antimicrobial susceptibility, and factors associated with Salmonella infection among patients who report gastrointestinal complaints. Methods: A cross-sectional study was conducted from December 2017 to February 2018 among patients with gastrointestinal complaints at Kibena Regional Hospital. Stool samples were submitted for isolation of Salmonella spp. Identification was based on conventional biochemical tests and serotyping to differentiate typhoid and nontyphoid Salmonella (NTS). Antimicrobial susceptibility was performed using the Kirby-Bauer disc diffusion method. Multivariable logistic regression analysis was performed to examine the factors independently associated with Salmonella infection. Results: The prevalence of Salmonella infection among participants with gastrointestinal complaints was 16.5% (95% CI: 12.7-21.1) of them, 83.7, 95% CI: 70.9-91.5 were NTS while 16.3, 95% CI: 8.5-29.0 were Typhoid Salmonella species. All isolates were sensitive to ceftriaxone and ciprofloxacin, whereas 27.8 and 100% were resistant to co-trimoxazole and ampicillin respectively. The odd of Salmonella infection was fourfold higher among participants with formal employment (AOR 3.8, 95% CI, 1.53-9.40). Use of water from wells/rivers (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 a restaurant (AOR 3.4, 95% CI, 1.28-8.93) had increased odds 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 independent symptoms that predict Salmonella infection. Conclusion: There is a high prevalence of Salmonella infection among people who report gastrointestinal complaints and it is clinically predicated by diarhoea and abdominal pain. Employed participants and those eating at restaurant and drinking unsafe water had higher risk of infection. Salmonella spp. causing gastroenteritis has developed resistance to commonly used antibiotics.