Urban transportation contributes significantly to CO₂ emissions. Public transport systems are a good strategy to reduce these, but the emissions generated by public transport vehicles should not be neglected during the design of the service. The Transit Network Design and Frequency Setting Problem (TNDFSP) has usually been addressed considering only the passengers’ and the operator's point of view. However, we show it is worthwhile to consider also the emissions already during this planning phase. This paper proposes a memetic algorithm to address the bi-objective TNDFSP where both the total travel time and the CO₂ emissions are minimized. The analysis considers a heterogeneous fleet, meaning that buses of different sizes and technologies can be assigned under a budget constraint. The results on benchmark instances show that the proposed memetic algorithm performs as well as state-of-the-art algorithms where CO₂ emissions are not considered. In addition, several experiments are carried out to observe the effect of incorporating emissions and heterogeneous fleet into the model. The heterogeneous fleet allows reducing travel times and emissions at the same time, compared to solutions without a heterogeneous fleet. Moreover, the explicit minimization of CO₂ emissions within a bi-objective framework allows illustrating the trade-off between both objectives. Reductions of about 30% in the emissions can be achieved by increasing the travel time only 1%, while the costs for the operator remain the same. This clearly demonstrates the benefits of considering both the CO₂ emissions and a heterogeneous fleet during the design stage of public transport systems.