The aim of this study was to evaluate the effects of ruminal and abomasum protein supplementation in cattle consuming tropical forages on nutritional characteristics, fibrolytic enzymatic activity, in vitro degradation of insoluble fiber, and the ruminal microbial community profile. Two experiments were sequentially conducted and differed only in the forage quality that was offered: medium-quality forage (MQF, 78.4 g of crude protein, CP/kg of dry matter, DM) and high-quality forage (HQF, 98.6 g CP/kg DM). Four Nellore young bulls fitted with rumen and abomasum cannulas were utilized, and the following supplementation schemes were evaluated: control (only forage), daily supply of 230 g of additional CP (casein) into the rumen, daily supply of 230 g of additional CP into the abomasum, and supply of 115 g of additional CP into the rumen and 115 g of additional CP into the abomasum. Experiments were conducted using a 4 × 4 Latin square design where two sequential squares were used, one for MQF and other for HQF. The improvement in forage quality increased (P < 0.05) intake, fiber digestibility, and nitrogen retention. Supplementation, independently of site (rumen, abomasum or both), increased (P < 0.05) nitrogen balance in the rumen and in the animal’s body. Ruminal supplementation increased (P < 0.05) rumen ammonia nitrogen concentration and consequently in vitro degradation rate of neutral detergent fiber. Enzymatic activity was not affected (P > 0.05) by forage quality. However, in disagreement with data of degradation and digestibility, decreases in enzymatic activity were observed mainly for post-ruminal supplementation. The improvement of forage quality increased (P < 0.05) the richness and diversity associated with liquid fractions from ruminal fluid, although there was no effect on solid fractions. The improvement in forage quality and protein supplementation increases nitrogen accretion in the animal body. Well-defined associations cannot be established between production characteristics and enzymatic activities, and rumen microbial diversity in cattle fed tropical forages and receiving protein supplementation.