BACKGROUND: Adherent-invasive Escherichia coli (AIEC) is linked to intestinal inflammation in inflammatory bowel disease (IBD). Arthrospira platensis and Lactobacillus helveticus exhibit anti-inflammatory properties individually, yet their effects remain underexplored in IBD-associated inflammation. We aimed to investigate the anti-inflammatory potential of L. helveticus and the hydroalcoholic extract of A. platensis (HA-A. platensis) in Caco-2 cells inflamed by IBD-associated E. coli. METHODS: Caco-2 cells inflamed by a Crohn's disease (CD)-associated E. coli strain (MOI 10) were treated with HA-A. platensis (2 mg/mL) and/or L. helveticus (MOI 50) in live (LBC), heat-killed (HKC), or cell-free supernatant (CFS) forms. The anti-invasion/adhesion properties of L. helveticus and/or HA-A. platensis were investigated by assessing the CD-associated E. coli invasion/adhesion rate (%). Signaling molecules (NF-kappaB, STAT3, NOD2) were analyzed via qPCR to capture pathway activation dynamics, while cytokines (TNF-alpha, IL-1beta, IL-8, IL-10) were quantified by ELISA to assess secreted functional proteins. RESULTS: HA-A. platensis reduced E. coli adhesion by 68% (P < 0.001) and completely inhibited invasion. L. helveticus (live form) decreased adhesion by 88% and invasion by 90%. Combined treatment showed synergistic effects, reducing adhesion by 89% and fully blocking invasion. HA-A. platensis downregulated STAT3 expression by 0.4-fold (P < 0.01), while L. helveticus (heat-killed form) reduced NF-kappaB by 0.51-fold (P < 0.05) and increased NOD2 by 1.8-fold (P < 0.01). Cytokine analysis revealed that HA-A. platensis decreased IL-1beta by 0.61-fold (P < 0.001), and L. helveticus (heat-killed) reduced TNF-alpha (0.51-fold) and IL-8 (0.23-fold) while elevating anti-inflammatory IL-10 (4.39-fold; P < 0.001). CONCLUSIONS: L. helveticus and HA-A. platensis synergistically inhibit CD-associated E. coli pathogenicity and modulate inflammatory responses in vitro. These findings highlight their potential as adjunctive therapies for CD, warranting further preclinical validation.