Agro-industrial by-products may provide alternative substrate constituents for short-cycle barley fodder production. Three olive pomace–sunflower hull–eggshell mixtures were assessed with Saida 183, Rihane 03 and El Fouara 97 barley in a 3 × 4 factorial design with three independent trays per combination. Fresh biomass from 36 individual trays averaged 580.2, 657.1, 776.9, 623.8 g/tray under control, M1, M2 and M3, respectively (n = 9 trays per treatment). Two-way ANOVA detected a substrate-treatment effect on fresh biomass (p < 0.001), whereas cultivar and interaction effects were not significant. Dry matter was independently determined three times per treatment (12 observations): 11.98 ± 0.04%, 12.23 ± 0.09%, 12.05 ± 0.07% and 11.88 ± 0.06%. Dry-matter percentage differed by treatment (one-way ANOVA F(3,8) = 14.35, p = 0.00139), with M1 above the other treatments in Tukey comparisons. Treatment-level dry-matter equivalents calculated from fresh-biomass means were 69.50, 80.36, 93.62, 74.10 g/tray. Biochemical factorial models identified cultivar associations, while some other findings were estimator-sensitive. Dry-matter equivalents should not be interpreted as separately measured yields, and analytical units require verification against original calibration records.The glucose, BSA and proline calibration standards are documented in Supplement S9. Biochemical comparisons in this study are reported primarily on the directly observed optical-density scale.