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Organic matter is a fundamental component of a substrate. However, different organic matter sources can provide different physiological responses from seedlings as a result of different types and concentrations of humic substances, which influence the functioning of the plant and the structuring of the soil. The objective of this work was to quantify photosynthetic pigments, gas exchange, and secondary metabolite indices in the abaxial and adaxial parts of leaves of Conilon coffee clonal plantlets grown in different sources of organic materials. The experiment was arranged in a randomized block design with five treatments and five replications. The treatments consisted of different sources of organic matter added to the substrate: 85% soil + 15% urban waste compost; 85% soil + 15% mature cattle manure; 85% soil + 15% dairy residue; 85% soil + 15% tannery sludge; and a Control: 100% soil, without addition of organic material. The contents of chlorophylls a, b, and total, carotenoids, and their ratios were analyzed by destructive methods. Indirect measurements of nitrogen and chlorophyll (SPAD) were performed by non-destructive methods. Fluorescence indices of flavonoids, anthocyanins, total chlorophyll, and nitrogen balance were estimated using a Multiplex® sensor. The substrate added with dairy residue provided the highest chlorophyll a production in the adaxial part, which was confirmed by the SPAD index. Tannery sludge provided the highest production of flavonoids. The comparison between the two forms of evaluation showed that the abaxial part of the leaf had greater sensitivity and that the Multiplex® sensor showed higher sensitivity for detection of indices. No changes were found in plant photosynthesis. However, greater stress was detected in the treatment with tannery sludge.