Rooting substrate and fertilization are key components in the production of containerized seedlings, as they can influence the morphological and physiological characteristics of the plants, which in turn can impact outplanting performance. The objective of the study was to evaluate the effect of four substrates based on mixtures of peat moss (PM), composted bark (CB) and raw pine sawdust (PS), combined with two doses of controlled release fertilizer (CRF) and one non-fertilized control, on the growth of Pinus cembroides Zucc. in the nursery. The treatments were: M1: 50+25+25, M2: 25+25+50, M3: 25+50+25 and M4: 50+50+0 of PM+ CB+ PS (% by volume), respectively. Fertilizer treatments used a controlled release fertilizer (Multicote®): F1: 3 kg m−3 and F2: 6 kg m−3 and a control (WF: with no added fertilizer). The treatments were distributed in a randomized complete block design, with a factorial arrangement of 4 × 3 and six replications. The variables evaluated were: height, seedling diameter, dry biomass, Dickson Quality Index, N, P and K content. Regardless of the substrate, the high fertilizer dose (6 kg m−3) improved most morphological variables. In addition, the high fertilizer dose resulted in foliar N, P and K concentrations within recommended ranges for all substrates. The substrate containing only peat moss and composted bark (M4 + F1 and M4 + F2) had the best growth response. However, the substrate composed of 25% peat moss, 50% composted bark and 25% raw pine sawdust with the high fertilizer dose (M3 + F2) resulted in acceptable seedling growth, and may be preferred if the cost of the substrate is a concern to nursery manager.