The objective of this study was to evaluate whether providing chitosan (CHI) to cows fed diets supplemented with whole raw soybeans (WRS) would affect the nutrient intake and digestibility, ruminal fermentation and bacterial populations, microbial protein synthesis, N utilization, blood metabolites, and milk yield and composition of dairy cows. Twenty-four multiparous Holstein cows (141 ± 37.1 d in milk, 38.8 ± 6.42 kg/d of milk yield; mean ± SD) were enrolled to a 4 × 4 Latin square design experiment with 23-d periods. Cows were blocked within Latin squares according to milk yield, days in milk, body weight, and rumen cannula (n = 8). A 2 × 2 factorial treatment arrangement was randomly assigned to cows within blocks. Treatments were composed of diets with 2 inclusion rates of WRS (0 or 14% diet dry matter) and 2 doses of CHI (0 or 4 g/kg of dry matter, Polymar Ciência e Nutrição, Fortaleza, Brazil). In general, CHI+WRS negatively affected nutrient intake and digestibility of cows, decreasing milk yield and solids production. The CHI increased ruminal pH and decreased acetate to propionate ratio, and WRS reduced NH3-N concentration and acetate to propionate in the rumen. The CHI reduced the relative bacterial population of Butyrivibrio group, whereas WRS decreased the relative bacterial population of Butyrivibrio group, and Fibrobacter succinogenes, and increased the relative bacterial population of Streptococcus bovis. No interaction effects between CHI and WRS were observed on ruminal fermentation and bacterial populations. The CHI+WRS decreased N intake, microbial N synthesis, and N secreted in milk of cows. The WRS increased N excreted in feces and consequently decreased the N excreted in urine. The CHI had no effects on blood metabolites, but WRS decreased blood concentrations of glucose and increased blood cholesterol concentration. The CHI and WRS improved efficiency of milk yield of cows in terms of fat-corrected milk, energy-corrected milk, and net energy of lactation. The CHI increased milk concentration [g/100 g of fatty acids (FA)] of 18:1 trans-11, 18:2 cis-9,cis-12, 18:3 cis-9,cis-12,cis-15, 18:1 cis-9,trans-11, total monounsaturated FA, and total polyunsaturated FA. The WRS increased total monounsaturated FA, polyunsaturated FA, and 18:0 to unsaturated FA ratio in milk of cows. Evidence indicates that supplementing diets with unsaturated fat sources along with CHI negatively affects nutrient intake and digestibility of cows, resulting in less milk production. Diet supplementation with CHI or WRS can improve feed efficiency and increases unsaturated FA concentration in milk of dairy cows.