Salmonella continues to be a significant contributor to global foodborne illnesses, and poultry products are frequently associated with outbreaks of salmonellosis. Removal of in-feed antibiotic growth promoters (AGPs) resulted in an increase in the incidence of coccidiosis and necrotic enteritis (NE) in poultry. The effects of co-infection of broilers with Salmonella (S) and subsequent exposure to Eimeria maxima (E) and Clostridium perfringens (CP) (to induce necrotic enteritis, NE) with or without dietary AGP supplementation in the feed on broiler growth performance, gut barrier integrity, microbiota and the virulence factors of CP isolates in commercial broilers raised under no-antibiotic-ever (NAE) and conventional (CV) poultry production systems was evaluated. Co-infection with Salmonella and subsequent challenges with E and CP (SNE) had significant adverse effect on broiler growth performance and increased Salmonella shedding. SNE also experienced more severe intestinal damage and heightened inflammation compared to infection with NE alone. Dietary supplementation of bacitracin methylene disalicylate (AGP) improved the performance and intestinal health of birds in non-challenge groups but did not yield any beneficial effects in birds exposed to mixed challenge (SNE+AGP; SNEA). NE, SNE, and SNEA caused dysbiosis in the cecum during the peak infection, marked by an increase in the Bacteroidetes phylum, Phocaeicola, and Clostridium genera, and a decrease in Firmicutes and Faecalibacterium genus in the cecum. The prevalence of CP in CV and NAE broiler farms and the genetic relationships between CP isolates from diseased and healthy chickens from both CV and NAE farms was evaluated. The prevalence of CP was higher in NAE broiler farms (44.1%) compared to CV farms (35.52%; P = 0.008). The netB and tpeL toxin genes were more common in CP isolates from diseased (NE) chickens than in isolates from healthy chickens (P <0.05). The presence of the cpb2 gene was similar between CP isolated from diseased birds and healthy birds (P >0.05). The C. perfringens netB and tpeL genes may play a role in the development of NE, but the role of cpb2 in NE disease induction is still unclear.