Abstract Marine prairies play various crucial roles in marine ecosystems. The seagrasses that compose them are one of the most important components engineering the marine coastal system, providing significant spatial niches. Some of the seagrasses found in marine prairies are protected, and it is not recommended to sample them with destructive methods. Non-destructive methods such as remote sensing have been proposed as important means of studying these protected species. In the present study, the acoustic scattering properties of Cymodocea nodosa were studied with two different in/ex situ experiments conducted on a Turkish Mediterranean coast using a scientific echosounder (206 kHz split beam transducer) in different months over the years 2011 and 2012. After a series of acoustic processes, correlations and regression equations were established between different acoustic parameters of the Elementary Distance Sampling Units and biometric traits of below/above ground parts of the seagrass. The relationships were logarithmically established producing first a Rayleigh zone, followed by a geometrical zone that occurred with increased biometrics. No seasonal difference occurred in the relationships for the above-ground parts. Unlike the leaves, seagrass sheaths demonstrated unstable echo energy, inconsistent relationships, and unexplained acoustic responses over the span of several months. Regarding leaf density changing in time, significant relationships were explained as a function of the acoustic zones. Four points were highlighted to explain the differences in the estimations between the two experiments; i) the backscattering strengths depended on strength of biomass and its fractions (leaf area, shoot density and volume) between the two experiments, ii) the first experiment measured backscattering strength from individual specimens, but the second experiment was performed on the total biomass of seagrass per unit area, iii) different frequency response to the biometrics occurred in the two experiments, and iv) the non-linear effect of the sheath could not be separated from that of the leaf during the second experiment. The present study was the first attempt to characterize relationships between the biometric and acoustic backscattering properties of C. nodosa, and will guide researchers in future use of non-destructive methods.