Background/significance Salmonella gastroenteritis causes significant morbidity among pediatric patients, mainly in developing world, such as the Middle East and North Africa (MENA) region. Concurrently, data from MENA countries like Iran, regarding prevalence of Salmonella serotypes, antimicrobial susceptibility, and biofilm production is scarce. Material & methods Slide agglutination was used to determine the serogroup of 140 Salmonella isolates recovered from 4477 stool specimens collected from children with gastroenteritis, and isolates were serotyped by PCR assay. The antimicrobial susceptibility of isolates to five first line drugs was assessed by disk diffusion assay using CLSI guidelines. Semi-quantitative evaluation of biofilm production was done by microtiter plate assay followed by PCR detection of biofilm-associated virulence genes csgD, pefA, and bcsA for each isolate. Results Nearly 94% of Salmonella isolates were recovered from less than or equai to 5-year-old patients, and 99% of isolates were non-typhoidal. While we found extensive diversity among Salmonella isolates, serogroup D (46%) predominated, and Salmonella Enteritidis (41%) was the most common serotype that showed the highest antimicrobial susceptibility rate (> 96%). For the first time in Iran, S. Newport serotype from human specimens was isolated. Most isolates were sensitive to all test antimicrobials, but 35% of isolates were not-typed (NT) that showed the highest resistance with 48% being resistant to greater than or equal to 1 test antimicrobial. Majority of isolates made weak (or no) biofilm, and we found a weak association between antimicrobial susceptibility, biofilm production, or virulence genes csgD, pefA, and bcsA. Conclusions The most effective measure that may control pediatric salmonellosis outbreaks is raising awareness of parents of preschoolers about food safety. Isolation of highly diverse Salmonella serotypes, including many commonly isolated from animals, indicates widespread contamination of the food chain. Majority of serotypes were sensitive to first-line antimicrobials, thus presently, pediatric Salmonella infections in this region may be controlled by conventional antimicrobials. However, despite the current trend, an imminent emergence of resistant Salmonella strains is foreseen, since various serotypes resistant to > 1 antimicrobial agent are typically associated with animals. Our results warrant further investigation that includes correlation analysis of clinical data regarding treatment outcomes, and serotype attributes like virulence genes. Keywords: Salmonella serogroups, Serotypes, Antimicrobial susceptibility, Biofilm, Virulence genes