The complement system may be activated in the posterior segment of the eye with chorioretinal disease, which may be reflected to the concentration of anaphylatoxins in the aqueous humor. Little is known about the distribution of anaphylatoxins in the aqueous and vitreous humor. The aim of the present study was to investigate the distribution of anaphylatoxin concentration in the aqueous and vitreous humor of the eyes with idiopathic epiretinal membrane or idiopathic macular hole. This was an experimental, observational case series. This study included 43 eyes from 43 patients; 29 eyes with idiopathic epiretinal membrane, and 14 eyes with idiopathic macular hole. All 43 eyes underwent cataract surgery and vitrectomy. The aqueous and vitreous humor were collected at the surgery. The anaphylatoxin concentrations were measured by using a cytometric beads array, and the respective C3a, C4a, and C5a concentrations were 2.003 ± 0.679 (mean ± standard deviation) ng/ml, 1.389 ± 0.419 ng/ml, and 0.003 ± 0.004 ng/ml in the aqueous humor, and 1.236 ± 0.642 ng/ml, 1.250 ± 0.542 ng/ml, and 0.048 ± 0.069 ng/ml in the vitreous humor. The mean C3a concentration in the aqueous humor was significantly higher than in the vitreous humor in 43 eyes of iMH and iERM (P < 0.001). The mean C4a concentration showed no significant difference between the aqueous humor and vitreous humor (P = 0.282), and the mean C5a in the aqueous humor was significantly lower than in the vitreous humor overall (P < 0.001). The C3a concentration in the aqueous humor strongly correlated with that in the vitreous humor (R = 0.510, P < 0.001). The concentrations of C4a and C5a in the aqueous humor moderately correlated with those in the vitreous humor (C4a; R = 0.356, P = 0.019, C5a; R = 0.464, P = 0.022). In conclusion, the anaphylatoxin concentrations measured by cytometric beads array in the aqueous humor may be associated with those measured in the vitreous humor.