The aim of this study was to evaluate the effect of human blood contamination, before and after hardening of the materials, on microhardness of high-viscosity Fuji IX GP Extra (Fuji IX) and resin-modified Fuji II LC (Fuji II) glass-ionomer cement (GIC) and glass-hybrid material EQUIA Forte HT (EQUIA), with and without protective coating EQUIA Forte Coat (Coat), before and after thermocycling. Four groups (n = 40): 1. Fuji IX; 2. Fuji II; 3. EQUIA and 4. EQUIA + Coat were further subdivided into 3 subgroups: (1) Control; (2) blood contamination before hardening; (3) blood contamination after hardening, resulting in a total of 12 groups of 10 samples each. Samples were prepared using teflon molds (5 mm × 2 mm). Microhardness was measured using a Vickers microhardness tester before and after thermocycling (10,000 cycles), and data were statistically analyzed (Kolmogorov–Smirnov test, ANOVA, Scheffe’s test). In the control groups, the highest microhardness was measured for EQUIA+Coat before thermocycling (70.71 ± 8.79) and after thermocycling (68.6 ± 7.65). Within the groups exposed to blood after hardening, the highest microhardness was recorded in the thermocycled EQUIA+Coat group (73.07 ± 8.85). Blood contamination before hardening negatively affected the microhardness of Fuji II, Fuji IX, and EQUIA+Coat. Exposure to blood after hardening increased the microhardness of Fuji IX and EQUIA, thermocycled Fuji IX and thermocycled EQUIA + Coat samples.