A novel 4-thiazolidinone derivative Les-6490 (pyrazol-4-thiazolidinone hybrid) was designed, synthesized, and characterized by spectral data. The compound was screened for its antimicrobial activity against some pathogenic bacteria and fungi and showed activity against Staphylococcus and Saccharomyces cerevisiae (the Minimum Inhibitory Concentration (MIC) 820 μM.). The compound was studied in the rat adjuvant arthritis model (Freund’s Adjuvant) in vivo. Parietal and fecal microbial composition using 16S rRNA metagenome sequences was checked. We employed a range of analytical techniques, including Taxonomic Profiling (Taxa Analysis), Diversity Metrics (Alpha and Beta Diversity Analysis), Multivariate Statistical Methods (Principal Coordinates Analysis, Principal Component Analysis, Non-Metric Multidimensional Scaling), Clustering Analysis (Unweighted Pair-group Method with Arithmetic Mean), and Comparative Statistical Approaches (Community Differences Analysis, Between Group Variation Analysis, Metastat Analysis). The compound significantly impacted an increasing level of anti-inflammatory microorganisms (Blautia, Faecalibacterium prausnitzii, Succivibrionaceae, and Coriobacteriales) relative recovery of fecal microbiota composition. Anti-Treponemal activity in vivo was also noted. The tested compound Les-6490 has potential prebiotic activity with an indirect anti-inflammatory effect.