Biomedical imaging technologies are designed to offer functional, anatomical, and molecular details related to the internal organs. Photoacoustic imaging (PAI) is becoming familiar among researchers and industrialists. The PAI is found useful in several applications of brain and cancer imaging such as prostate cancer, breast cancer, and ovarian cancer. At the same time, the vessel images hold important medical details which offer strategies for a qualified diagnosis. Recently developed image processing techniques can be employed to segment vessels. Since vessel segmentation on PAI is a difficult process, this paper employs metaheuristic optimization-based vascular segmentation techniques for PAI. The proposed model involves two distinct kinds of vessel segmentation approaches such as Shannon’s entropy function (SEF) and multilevel Otsu thresholding (MLOT). Moreover, the threshold value and entropy function in the segmentation process are optimized using three metaheuristics such as the cuckoo search (CS), equilibrium optimizer (EO), and harmony search (HS) algorithms. A detailed experimental analysis is made on benchmark PAI dataset, and the results are inspected under varying aspects. The obtained results pointed out the supremacy of the presented model with a higher accuracy of 98.71%.