This pilot study aimed to establish the interrelationship between collagen and mast cells in periapical granulomas and periapical cysts. An observational cross-sectional study was conducted on the paraffin-embedded tissue sections of 68 specimens (34 periapical granulomas and 34 periapical cysts). The specimens were stained with picrosirius to observe collagen fiber birefringence and anti-tryptase antibody to evaluate the mast cell count immunohistochemically. The mean number and birefringence of collagen fibers, as well as the mean number of mast cells (total, granulated, and degranulated), and the mean inflammatory cell density were calculated. The data obtained were analyzed using the Kruskal Wallis test, Mann Whitney test, and Spearman correlation test ( < 0.05). The mean number of thick collagen fibers was higher in periapical cysts, while that of thin fibers was higher in granulomas ( = 0.00). Cysts emitted orange-yellow to red birefringence, whereas periapical granulomas had predominantly green fibers ( = 0.00). The mean inflammatory cell density was comparable in all groups ( = 0.129). The number of total, degranulated, and granulated mast cells exhibited significant results ( = 0.00) in both groups. Thick cyst fibers showed significant inverse correlations with inflammation and degranulated mast cells ( = 0.041, 0.04 respectively). Mast cells and inflammatory cells influenced the nature of collagen fiber formation and its birefringence. This finding may assist in the prediction of the nature, pathogenesis, and biological behavior of periapical lesions.