Introduction The diagnosis and assessment of apical periodontitis by traditional periapical radiographs can be challenging and might yield false-negative results. The aim of this study was to determine whether interleukin-1beta (IL-1β) and dentin sialoprotein (DSP) in gingival crevicular fluid (GCF) can be used as biological markers for apical periodontitis. Methods Forty healthy patients with teeth diagnosed with apical periodontitis of pulpal origin were included in the study. GCF samples were obtained from the diseased tooth and from a healthy contralateral control tooth. Total protein concentration in each sample was determined by using the Bio-Rad protein assay. Enzyme-linked immunosorbent assay was used to analyze the concentration of IL-1β and DSP in the samples. Results Protein content of the GCF was statistically significantly higher in the disease group compared with the control group. The levels of IL-1β and DSP were not statistically different between disease and control groups. Conclusions Although this study was unable to demonstrate a significantly higher level of IL-1β or DSP in the GCF of teeth with apical periodontitis, the observed presence of a significantly higher level of total protein in the GCF of diseased teeth suggests the possible role of total protein level as a marker for periapical disease.