Early blight disease of tomato is one of the most devastating biotic stresses worldwide, and in Iran, Alternaria alternata is one of the most predominant species causing the disease. In the current study, a diverse collection of 35 tomato genotypes and implication of 5 SlWRKYs and 7 PR genes as well as enzymatic activity were evaluated on resistant and susceptible cultivars through real-time polymerase chain reaction at transplanting and maturing stages and by measuring product formation using spectrophotometry. The results indicated that the expression of these antifungal genes in 14 genotypes at two growth stages after inoculation with A. alternata highly enhanced by 1–50-fold. There was also significant upregulation of WRKYs and PRs genes among the resistant tomato varieties in comparison to susceptible and control varieties at both stages. These findings demonstrate the varieties that showed increased or decreased SlWRKY1 expression also displayed similar changes in the expression of PR1 and PR2 genes. Furthermore, the differential expression patterns of SlWRKY1 and SlWRKY11 were consistent with PR7 and PDF1.2 expression patterns. The analysis of enzymatic activity of PR2 and PR3 proteins, β-1,3-glucanase, and chitinase showed the highest level of activity in resistant inoculated genotypes against A. alternata. Therefore, the current findings suggest the possible involvement of these transcription factors in the increased expression of PR genes in response to A. alternata infection. •Some tomato varieties are resistant to early blight disease caused by A. alternata.•The expression of defense-related genes provides EB disease resistance.•Chitinase and glucanase contribute to resistance to EB disease in tomato.•Expression and enzymatic activity of chitinase and glucanase are directly related.