This paper presents an inverse finite element (FE) approach aimed at estimating multi-layered human penile tissues. The inverse FE approach integrates experimental force–displacement and boundary deformation data of penile tissues with a developed FE model and uses new experimental data on human penile tissue. The experimental study encompasses whole organ plate-compression tests and individual layer tensile and compression tests, providing comprehensive insights into the tissue's mechanical behaviour. The biomechanical characterisation of penile tissue is of crucial significance for understanding its mechanical behaviour under various physiological and pathological conditions. The FE model is constructed using the realistic geometry of the penile segment and appropriate constitutive models for each tissue layer to leverage the accuracy and consistency of the model. Through systematic variation of tissue parameters in the inverse FE algorithm, simulations achieve the best match with both force–displacement and deformed boundary results obtained from the whole organ plate-compression tests. Test results from individual tissue layers are also utilised to assess the estimated parameters. The proposed inverse FE approach allows for the estimation of penile tissue parameters with high precision and reliability, shedding light on the mechanical properties of this complex biological organ. This work has applications not only in urology but also for researchers in various disciplines of biomechanics. As a result, our study contributes to advancing the understanding of human penile tissue mechanics whilst the methodology could also be applied to a range of other soft biological tissues. Statement of significanceThis research uses a multi-target inverse finite element (FE) approach for estimating the material parameters of human penile tissues. By integrating experimental data and a realistic FE model, this study achieves high-precision constitutive model parameter estimation, offering key insights into penile tissue mechanics under various loading conditions. The significance of this work lies in the use of this inverse FE approach for fresh-frozen human penile tissues, to identify the mechanical properties and constitutive models for both segregated tunica albuginea and corpus cavernosum as well as intact penile tissue segments. The study's scientific impact lies in its advancement of the understanding of human urological tissue mechanics, impacting researchers and clinicians alike.