Clostridioides difficile infection (CDI), along with the severity of the disease, is intimately tied to the production of bacterial toxins. The expression of toxins is governed by intricate mechanisms that respond to various intra- and intercellular stimuli. Recent genomic studies have identified an accessory gene regulator (agr) system in C. difficile, similar to that found in Staphylococcus aureus, playing a pivotal role in coordinating toxin synthesis. Nevertheless, the agr system of tcdA(+)B(+) C. difficile clinical isolates, particularly in terms of phylogenetic analysis, phenotypic characteristics, and virulence expression, has not been extensively studied in Iranian isolates. This investigation aimed to characterize agr type and agrD sequences in tcdA(+)B(+) clinical isolates obtained from Iranian diarrheal patients over a two-year period, utilizing specifically designed primer sets. Basal expression levels of virulence and regulatory factors, genotyping, sporulation efficiency, and motility were also examined. PCR analysis of 50 tcdA(+)B(+) C. difficile isolates revealed universal presence of agr1, with 44 (88 %) isolates also possessing agr2. A notable number of isolates (n = 12, 24 %) exhibited a threonine (T) to lysine (K) substitution at position 47 (T47K) in the AgrD1 C-terminal charged region, predominantly associated with RT126/tcdC-A/toxinotype V genotype. The AgrD2 sequence remained conserved across 44 isolates, aligning with R20291 strain. The study uncovered significant variability in sporulation efficiency among isolates, with high efficiency correlating with tcdC-A genotype. Furthermore, 70 % of isolates demonstrated motility, with 58 % showing high motility in semi-solid brain heart infusion broth medium. Our findings highlight widespread prevalence of agr1 and the previously unrecognized T47K substitution within agr1, along with the distribution of two agr loci in tcdA(+)B(+) C. difficile isolates. This study also provides novel phenotypic insights into tcdA(+)B(+) C. difficile isolates from Iranian patients, paving the way for more comprehensive functional and clinical studies of agr loci.