This study aimed to assess the potential of using spent white button mushroom substrate (SMS) as replacement for peat for producing cherry tomato seedlings, considering different proportions of fresh and composted SMS. Phase I focused on testing the performance of seedlings raised in different growing media mixes and Phase II on the assessment of the subsequent yields of the tomatoes raised in selected treatments. Results showed that the use of fresh SMS as a replacement for the peat-based control substrate was limited to 20% (% w/v), due to its high salinity. However, the control substrate could be replaced completely with composted SMS (Composted SMS 100) without any significant differences in the growth of the seedlings. Based on the most promising results from Phase I, yields of plants raised in composted SMS (Composted SMS 40 and 100), fresh SMS (Fresh SMS 20), and peat-based control were compared. There were no significant differences in the total yield (mass) between the treatments, but the average total number of fruits of the five harvest dates was higher in the Composted SMS 100 and in the control than in the other treatments. Notably, Composted SMS 100 and control produced greater mass of tomatoes at the final harvest. In conclusion, SMS provided an effective alternative for production of cherry tomato seedlings, with composting of the SMS before use being a crucial step. It was recommended that no more than 20% fresh SMS should be used to replace peat to ensure optimal growth of cherry tomato seedlings.