This study was carried out to determine the effects of addition of humate, probiotic, and their combination into diets on performance, egg quality, and yolk fatty acid composition of hens during the second laying period. Lohmann LSL white layers (n = 192), 46 weeks of age, were randomly divided into 4 groups and fed with basal diet (control, C), 0.3% humate (H), 0.3% probiotic (P), 0.15% humate + 0.15% probiotic (HP) for 18 weeks. Feed consumption and egg production were determined daily, egg weight was measured biweekly, and body weights were recorded at the beginning and the end of the experiment. Also, 12 egg samples from each group were randomly collected to determine the egg quality every 30 days. Laying performance, yolk color, and fatty acid composition were significantly (P < 0.05 and P < 0.01) affected by addition of humate, probiotic, and their combination into diets of layers. The HP group had higher cracked egg yield and feed conversion ratio values than control and H and P groups. Except for egg yolk color, the other egg quality parameters such as shape index, shell strength, shell thickness, albumen index, yolk index, and Haught unit were not affected by treatment (P < 0.05 and P < 0.01). The egg yolks of treatment groups had less stearic acid than those of control group. In conclusion, supplementation of humate and probiotic into the diets of laying hens increased monounsaturated fatty acids in yolk and improved feed conversion ratio and egg yolk color.